IGEM:Groningen/Notebook/iGEM 2011/2011/09/02: Difference between revisions
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== | ==2-9-11== | ||
<br> | |||
<br> Plasmid Prep of overnight cultures: cI-LVA and LasR-LVA in chloramphenicol vector+ K077002 and K07004 for cI production. | |||
<br> According to the ND1000- Nanodrop, DNA concentration was: around 30ng/μl | |||
<br> | |||
<br> Use the cI-LVA and LasR-LVA vectors for digestion | |||
<br> | |||
<br> Digestion: | |||
<br> | |||
<br> PBAD | |||
<br> 5.6 μl PBAD PCR product | |||
<br> 1μl EcoRI | |||
<br> 1μl SpeI | |||
<br> 2μl FD buffer | |||
<br> 10.4μl MQ water | |||
<br> | |||
<br> cI-LVA vector: | |||
<br> 4μl cI-LVA vector | |||
<br> 1μl EcoRI | |||
<br> 1μl XbaI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 20μl MQ water | |||
<br> | |||
<br> LasR-LVA vector: | |||
<br> 4μl LasR-LVA vector | |||
<br> 1μl EcoRI | |||
<br> 1μl XbaI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 20μl MQ water | |||
<br> | |||
<br> pSB1C3-DT vector: | |||
<br> 3μl pSB1C3-DT | |||
<br> 1μl EcoRI | |||
<br> 1μl XbaI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 21μl MQ water | |||
<br> | |||
<br> pSB1C3-DT vector | |||
<br> 3μl pSB1C3-DT | |||
<br> 1μl XbaI | |||
<br> 1μl PstI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 21μl MQ water | |||
<br> | |||
<br> LasR-LVA PCR product: | |||
<br> 4μl LasR-LVA PCR product | |||
<br> 1μl XbaI | |||
<br> 1μl PstI | |||
<br> 2μl FD buffer | |||
<br> 12μl MQ water | |||
<br> | |||
<br> Incubate the samples for 1h at 37°C | |||
<br> | |||
<br> After digestion: DNA clean up with the High Pure PCR Purification kit of Roche | |||
<br> | |||
<br> Ligation: | |||
<br> | |||
<br> PBAD-cI-LVA | |||
<br> 5μl PBAD | |||
<br> 8.5μl cI-LVA vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 3.5μl MQ water | |||
<br> | |||
<br> PBAD-LasR-LVA | |||
<br> 5μl PBAD | |||
<br> 8.5μl LasR-LVA vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 3.5μl MQ water | |||
<br> | |||
<br> PBAD-pSB1C3DT | |||
<br> 7μl PBAD | |||
<br> 8.5μl pSB1C3DT vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 1.5μl MQ water | |||
<br> | |||
<br> LasR-LVA in pSB1C3DT vector | |||
<br> 8.5μl LasR-LVA PCR product | |||
<br> 8.5μl pSB1C3DT vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> | |||
<br> Ligate for 30-40 minutes at room temperature | |||
<br> | |||
<br> Transformation: | |||
<br> Add to 40μl competent E.coli DH5alha cells 10μl ligation mixture | |||
<br> Incubate the samples for 30 min. on ice | |||
<br> Heat shock: | |||
<br> Incubate the samples for 45s at 42°C | |||
<br> After the heat shock: place the cells for 2 min on ice and add after the incubation time on ice 1ml of LB+25mM glucose. | |||
<br> Incubate the samples for 1h to 1.5 h at 37°C | |||
<br> Plate the cells out: Centrifuge the sample, leave 100μl of LB+glucose in the tube and resuspend the pellet. | |||
<br> Plate 90μl and 10μl of the resuspended pellet on plates containing antibiotic(s) | |||
<br> | |||
<br> Colony PCR | |||
<br> Colony PCR of the RBS-GFP-DT plasmid from the distribution kit. Yesterday, that sample had been transformed again, since there <br> were some difficulties with the plasmid in cloning purposes. It did not seem to have the insert (RBS-GFP-DT) anymore. | |||
<br> | |||
<br> PCR mix: | |||
<br> 10× Taq buffer: 20μl | |||
<br> dNTPs 10mM each: 4μl | |||
<br> MgCl2: 12μl | |||
<br> BB forward primer: 4μl | |||
<br> BB reverse primer: 4μl | |||
<br> Taq polymerase: 1μl | |||
<br> MQ water: 155μl | |||
<br> | |||
<br> PCR conditions: | |||
<br> Pre heated lid at 111°C | |||
<br> Denaturation: 94°C for 10 min | |||
<br> Cycle 33× | |||
<br> Denaturation: 94°C for 30s | |||
<br> Annealing: 60°C for 30s | |||
<br> Extension: 72°C for 2.5 min | |||
<br> Final extension: 72°C for 10 min | |||
<br> Store at 4°C infinite | |||
<br> | |||
<br> Analyse on a 1% TBE agarose gel | |||
<br> | |||
Revision as of 01:55, 2 September 2011
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